Our primary objective is to determine the amino acid sequence of tetanus toxin. We have purified tetanus toxin from culture filtrates and by extraction from cells of Clostridium tetani, Massachusetts strain. We have separated the toxin into its heavy and light chain components subsequent to reductive cleavage of its disulfide bonds. Each large peptide will be fragmented by cleavage with cyanogen bromide. This should produce about fifteen peptides from the heavy chain and about ten peptides from the light chain. The peptides will also be fragmented by trypsin which should yield an appreciably larger number of peptides than cyanogen bromide. The peptides from each fragmentation will be separated and sequenced. If a peptide is too large for overlap, other enzymes will be used to produce subpeptides. This project is well underway and we already have the partial sequence of one large peptide. We are also producing purified toxin in large quantities which are adequate for sequence work. These experiments should allow us to compare the amino acid sequence of this unusual protein with its secondary structure and that of other nontoxic soluble proteins. It should also increase our knowledge of the molecular mechanism of the pathology of tetanus and ultimately aid in the prevention of tetanus and care of patients afflicted by the disease.